Project Summary The epicardium covers the heart as a single epithelial layer, which provides extracellular matrix support and an active stem and progenitor cell pool. Epicardial embryonic development involves the dynamic motility and differentiation of epicardial derived progenitor cells (EPDCs), which facilitate the development of vascular smooth muscle and endothelial cells and fibroblasts in the heart. Migration and lineage specification of EPDCs is regulated by myocardin related transcription factors, also known as MRTF-A and MRTF-B, to promote myofibroblast formation and pericytic functions. More recently, our laboratory has published that MRTF-A and B deletion from the epicardial cell population with a WT1-driven Cre (called MRTFeDKO) impairs migration of EPDCs resulting in severe vascular deficiencies. Studies from our laboratory and others provide evidence that epicardial cell activation is critical for proper embryonic heart growth and maintenance. However, the contribution of EPDCs during adult cardiac remodeling remains unexplored. Cardiac repair after adult MI is greatly limited due to the inability of cardiomyocytes to divide and modest contribution of endogenous cardiac progenitor cells stemming from the epicardium. In order to investigate EPDC function during an adult setting of MI, acute myocardial infarction (MI) was induced in MRTFeDKO mice. Surprisingly, MRTFeDKO mice displayed a profound cardioprotective phenotype characterized by increased cardiac function and reduced fibrotic scarring compared to wildtype controls. We surmised that this phenotype was most likely due to alterations in the balance of EPDC maintenance and differentiation in MRTFeDKO mice. In this proposal, we will test if MRTF deletion in the epicardium counteracts negative remodeling of the heart by influencing subepicardial progenitor mobilization and cardiomyocyte function following MI. In order to identify novel underlying mechanisms that contribute to EPDC activity during cardiac regeneration following deletion of MRTFs, we performed a genome wide RNA-sequencing screen. Results of the RNA-sequencing revealed that neuronal guidance genes are highly enriched in EPDCs. Interestingly, neuronal guidance genes were inversely expressed in EPDCs lacking MRTFs. Axonal guidance genes are critical for neovascularization and proper heart contractility, however, there are currently no reports describing influence of cardiac innervation on EPDC function. Overall, accomplishing the stated aims of this proposal will yield a comprehensive characterization of EPDCs as it relates to MRTF signaling and axonal guidance cues. Specific Aims are: 1) Determine the role of MRTFs in post MI remodeling, 2) Determine the cellular mechanisms contributing to cardioprotection in MRTFeDKO and 3) Define the function of a novel epicardium-based axon guidance center in cardiac repair. These studies will describe the ability to harness a novel epicardium-based axon guidance-signaling center to improve cardiac repair and regeneration after injury.